Immunohistochemical detection of the human major vault protein LRP with two monoclonal antibodies in formalin-fixed, paraffin-embedded tissues.
Multidrug resistant cancer cells frequently overexpress the 110-kd lung resistance-related protein (LRP) as detected with the monoclonal antibody (MAb) LRP-56. Recently, we identified LRP as the major vault protein (MVP), which is the major constituent of vaults, multisubunit cellular organelles. Clinically, LRP/MVP expression in cancer at time of diagnosis provided a strong and independent prognostic factor for response to chemotherapy and outcome in different tumor types, notably acute myeloid leukemia and ovarian cancer. To facilitate additional immunohistopathological studies, we have optimized LRP/MVP detection in paraffin-embedded tissues using two monoclonal antibodies, LRP-56 and LMR-5. Blocking experiments showed that LRP-56 and LMR-5 MAbs detect different epitopes of LRP/MVP. Immunohistochemical studies with both MAbs in a panel of human multidrug resistant tumor cell lines, normal tissues, and colorectal tumors showed that LRP/MVP expression can be reliably detected after formalin-fixation and paraffm-embedding using overnight incubation at 4 degrees C with the primary MAbs at 5- to 10-fold higher concentrations (ie, 1 to 10 microg/ml) as currently used with frozen sections. Both streptavidin-biotin complex and alkaline phosphatase-anti-alkaline phosphatase techniques could be successfully used for signal-amplification. Staining quality did not benefit from antigen-retrieval pretreatments. The optimized staining methodology facilitates studies in archival material on the putative role of LRP/MVP in clinical drug resistance.
Schroeijers
A B
AB
Department of Pathology, Free University Hospital, Amsterdam, The Netherlands.
Scheffer
G L
GL
Flens
M J
MJ
Meijer
G A
GA
Izquierdo
M A
MA
van der Valk
P
P
Scheper
R J
RJ
D000911:Antibodies, Monoclonal; D018432:Drug Resistance, Multiple / Q000502:physiology; D005404:Fixatives; D005557:Formaldehyde; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods*; D009363:Neoplasm Proteins / Q000032:analysis*; D009369:Neoplasms / Q000737:chemistry; D016612:Paraffin Embedding; D012016:Reference Values; D014407:Tumor Cells, Cultured / Q000737:chemistry; D020394:Vault Ribonucleoprotein Particles; D000099187:Major Vault Protein
D000911:Antibodies, Monoclonal; D005404:Fixatives; D005557:Formaldehyde; D009363:Neoplasm Proteins; D020394:Vault Ribonucleoprotein Particles; D000099187:Major Vault Protein